The human disease hemochromatosis is a genetic disorder caused by excess uptake of iron from the diet, resulting in multi-organ failure and, often, liver cancer. Although a candidate gene for this disorder was recently identified, the molecular events through which iron causes damage to cells are largely unknown. Hepatic iron overload is frequently associated with chronic hepatitis and other inflammatory diseases of the liver. Similarly, the molecular events leading to iron accumulation are unknown. We have designed a genetic model of iron overload in S. cerevisiae by using a dominant mutant allele of the iron-sensing transcription factor AFT1. Expression of this dominant mutant allele, AFT1-1up, results in constitutive uptake of iron regardless of intracellular or extracellular iron abundance.Our research involves the use of whole-genome approaches to identify genes that are involved in iron metabolism in S. cerevisiae. In collaboration with the deparment of Genetics at Stanford University, we have employed DNA microarray analysis to identify over genes that are regulated by the transcription factor AFT1. All of the genes that were previously identified as being regulated by iron and AFT1 were identified with this technique, some characterized genes were unexpectedly found to be regulated by AFT1, and, in addition, many previously uncharacterized genes were identified, including six new putative transporters. Using available genome and protein databases, these newly identified genes have been grouped into families and experiments are underway to evaluate the functions of these genesOur studies of these newly identified genes have uncovered a novel pathway of siderophore-mediated iron uptake in yeast and we have identified the specific transporter for the therapeutic iron chelator desferrioxamine. Our data indicate that siderophore-mediated iron uptake occurs through an endosomal pathway not shared by the high-affinity transport system. We have also determined that iron uptake and nitrogen utilization are co-regulated processes in yeast and the mechanism of this regulation is being studied. These data were presented at the combined meetings of the International Association for the study of Disorders of Iron Metabolism and the International Conference on Proteins of Iron Metabolism in May, 1999 as well as in seminars on the NIH campus. Using this approach we hope to identify mammalian homologues of these proteins, and to define pathways of iron uptake, utilization, and detoxification in humans. This work will enable the design of rational therapeutic or preventative treatments for patients susceptible to iron overload. - iron overload yeast nitrogen metabolism siderophore hemochromatosis